Development of a bioassay guided genome mining approach for antifungal natural product discovery from pseudomonads

Zymoseptoria triticicauses Septoria Leaf Blotch disease of wheat and has evolved to overcome most chemical and genetic control methods. As such, new tools are required for future disease control.

We identifiedPseudomonasisolates that antagoniseZ. triticithrough the production of secreted secondary metabolites, using a novelin vitro Z. triticiantagonism assay. In addition to high-throughput qualitative assessment ofPseudomonasantagonism ofZ. tritici, a quantitative assessment identified variation in the sensitivity ofZ. triticiisolates to antagonism by a subset ofPseudomonasisolates.

Genome assemblies of 3 strongly antagonisticPseudomonasisolates were found to contain a predicted Biosynthetic Gene Cluster (BGC) with high sequence similarity to a reference BGC encoding the biosynthesis of the known antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG).

Mutagenesis of the core biosynthetic genephlDresulted in a loss of 2,4-DAPG production inPseudomonasisolate Roth82, and a loss ofZ. triticiinhibition in the antagonism assay. These results demonstrate that the describedin vitroantagonism assay can be used to identify, quantify and mechanistically characterise bacterial antagonism ofZ. triticithrough the production of secondary metabolites.

This is the first study to find significant differences in the response of genetically diverse isolates ofZ. triticiisolates to bacterial antagonists, suggesting sensitivity to bacterial antagonism exists as a quantitative trait within naturalZ. triticipopulations. Our approach can be used to identify and characterise putatively novel BGCs that encode natural products with antifungal activity againstZ. tritici.